Selective non-Watson-Crick pairing-guided DNA synthesis for locus-specific analysis of 8-oxo-7,8-dihydroguanine in telomeres at ultrahigh resolution

8-Oxo-7,8-dihydroguanine (OG) is the most common oxidative modification in chromosomal DNA induced by reactive oxygen species (ROS). OG damage promotes telomere shortening and dysfunction, contributing to numerous degenerative/aging-related diseases and cancers. However, few methods are available for locusspecific quantification of OG in telomeres because of trace level of OG in DNA and low sensitivity of existing assays. Herein, we demonstrate selective non-Watson-Crick pairing-guided DNA synthesis for locus-specific analysis of OG in telomeres at ultrahigh resolution. When target telomere-OG is present, it hybridizes with the padlock probe to generate a one-base gap opposite OG. Deoxyadenosine triphosphate (dATP) is then incorporated into the gap by Bsu DNA polymerase (Bsu Pol) to mediate single-base extension. Through toeholdmediated strand displacement (TMSD), the primer probe displaces telomere-OG, and then it hybridizes with the extended padlock probe to induce proximity ligation-dependent cyclization and subsequent RCA with the aid of Taq ligase and phi29 polymerase, producing numerous ssDNAs that can bind with SYBR Green II to generate a significant fluorescence signal. This method can detect telomere-OG with a limit of detection (LOD) of 3.50 x 10-18 M in vitro and 1.38 x 10-3 ng in vivo under hydrogen peroxide (H2O2) stress. Moreover, it can discriminate as little as 0.001 % telomere-OG in excessive undamaged DNAs, and quantify the OG level in telomere DNAs from diverse human cell lines under oxidative stresses. Notably, this method can be rapidly implemented in one tube under isothermal conditions, without any labeled probes, complex steps, and high sample consumption, facilitating oxidative damage-related biomedical research and clinical diagnosis.